Heretofore, coenzymes have been frequently used for chemical reagents for biochemical clinical investigations. Coenzymes, which have been used, include oxidation or reduction type nicotinamide adenine dinucleotide (NADH or NAD) or nicotinamide adenine dinucleotide phosphate (NADPH or NADP). However, those coenzymes have a problem with stability. The coenzyme changes to an oxidized coenzyme, NAD or NADP, by a very small amount of an enzyme included in an assay reagent, or disintegrates ADP ribose and others, thereby resulting in a change in structure which can be used by an enzyme. Therefore, there is a problem in that such a change leads to a decrease in total amount of the coenzyme and then lowers the sensitivity of the assay agent (Non-patent Document 1). For solving this problem, it is known that a coenzyme is preserved under temperature conditions as low as possible, such as under freezer storage or cold storage and a coenzyme-containing reagent is then freeze-dried. Alternatively, it is known that, as stabilizers for preserving the coenzyme in a state of solution, there are used, for example, an amine base, sodium hydroxide, a chelating agent, azide, boric acid, an alkali metal, an ammonium bicarbonate buffer, and an active-oxygen removing substance. Any of these methods has been conceived with the intention of preventing the coenzyme from being decomposed as described above to avoid a decrease in sensitivity (Patent Document 1 and Patent Document 2).
Coenzymes are used for immunologic clinical diagnostic reagents. For instance, in an enzymatic immunoassay, there has been known a coenzyme-cycling method as a method of detecting an alkaline-phosphatase (ALP)-labeled antibody with a phosphorylated coenzyme, NADP or NADPH, which is used as a substrate (Patent Document 3). Detection methods using enzyme-cycling reagents made of NADP or NADPH, alcohol dehydrogenase, diaphorase, alcohol, and tetrazolium salt have been well known in the art. For example, when NADP is used as a substrate for ALP, NAD is produced from NADP by ALP and the resulting NAD is then converted into NADP by alcohol dehydrogenase and alcohol. Subsequently, NADH returns to NAD by diaphorase and tetrazolium salt and the reaction of converting NAD to NADH then proceeds again. In this way, through the cyclic reaction between NAD and NADH, formazan dyes produced from tetrazolium salt accumulate in a reaction solution. Thus, the amount of the dyes is measured to determine the activity of ALP.
Even those reagents also have problems with stability of coenzyme, like biochemical reagents, for prolonged preservation of the reagents, coenzymes have been preserved in a free-dried state under temperature conditions as low as possible, such as under freezer storage or cold storage. In addition, an attempt has been made by selecting an appropriate buffer for stabilization.
In contrast, in recent years, simple assays, which are typified by assay reagents for influenza antigen and capable of obtaining assay results on the spot in a medical office or at bedside, have been becoming popular. Any of these point-of-care (POC) test reagents requires, if it should be preserved under cold storage or preservation of lower temperature, a specific device, such as a refrigerator or a freezer, and the preservation space for the reagent is also limited, so there is a problem in that a large amount of the reagent cannot be purchased and preserved. Therefore, a reagent, which can be preserved at room temperature, has been demanded. However, in these POC test reagents, for providing a reagent using a coenzyme for practical use, which can be preserved at room temperature, a prolonged preservation stability under severe conditions than the conventional refrigerating preservation has been already difficult as described above and now such a preservation stability should be attained under more severe conditions. Therefore, the practical use of a test reagent using a coenzyme in this field has been extremely difficult.
In addition, in the field of test reagents, in order to measure a very small amount of a substance of interest, it is demanded to enhance the sensitivity of the measurement. ALP has been well known as a target enzyme generally used in an enzymatic immunoassay, so a procedure of detecting ALP at high sensitivity has been demanded. In addition, the technique of enhancing the sensitivity is useful not only for microassay but also for reduction in amount of a sample or shortening the measuring time. Thus, this is the most desired technique in this field.    [Patent Document 1] JP 3470099 B    [Patent Document 2] JP 2001-61498 A    [Patent Document 3] JP 06-303996 A    [Non-patent Document 1] Basic experimental method for protein and enzyme, Nankodo Co., Ltd., page 426